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標題: Expression of Trigonopsis variabilis D-amino acid oxidase in transgenic rice for cephalosporin production
作者: Lin, S.Y.
Wang, J.D.
Lin, J.H.
關鍵字: Cephalosporin;D-amino acid oxidase;Japonica rice cultivar Taiken 9;Transgenic rice plant;Trigonopsis variabilis;t-cell epitopes;phosphoenolpyruvate carboxylase;escherichia-coli;mediated transformation;protein-production;plants;gene;maize;substitution;promoters
Project: Botanical Studies
期刊/報告no:: Botanical Studies, Volume 50, Issue 2, Page(s) 181-192.
Transgenic plants have become all effective system to produce recombinant proteins, and there are many examples of transgenic plants that successfully produce functional proteins. In this study, the japonica rice cultivar Taiken 9 was transformed through an Agrobacterium-mediated method to express D-amino acid oxidase (DAAO) from Trigonopsis variabilis. DAAO is a flavoenzyme that catalyzes the oxidation of cephalosporin C to produce the precursor of the cephalosporin antibiotic glutaryl-7-aminocephalosporin acid (G1-7-ACA). DAAO derived from T variabilis has the highest catalytic activity for cephalosporin C oxidation of DAAO enzymes that have been characterized. Trigonopsis daao was expressed in rice under the control of either the rice actin I (Act1) or maize phosphoenolpyruvate carboxylase (PEPC) promoter. Southern blot analysis demonstrated the integration of Trigonopsis daao gene into the rice genome. Furthermore, northern blot and western blot analysis demonstrated production of the daao transcript and accumulation of its protein in various tissues of transgenic rice plants using either the Act1 or PEPC promoter as compared with the wild type. DAAO activity was detected in both transgenic rice lines with a maximum specific activity of 65.5 +/- 7.4 U mg protein(-1) min(-1) detected in the leaves of transgenic plants containing the rice Act1 promoter. The transgenic rice plant with the rice Act1 promoter exhibited several fold higher DAAO activity than the plant with the maize PEPC promoter: 5.3- and 3.7-fold higher in the leaves and sheaths, respectively. No DAAO activity was detected in the grains of transgenic rice containing the PEPC promoter. Taken together, these results demonstrate that Trigonopsis daao is stably integrated into the transgenic rice genome, transcribed efficiently, and translated into a functional protein.
ISSN: 1817-406X
Appears in Collections:期刊論文

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